Branching morphogenesis is a dynamic process used to construct a variety of organs, such as kidneys, salivary glands, and the mammary glands. To study branching morphogenesis, we have been able to recapitulate in vivo tissue structures in three-dimensional (3D) engineered tissue arrays that resemble the structure of the branching mammary glands in vivo. Engineering branched tissue structures that display appropriate spatial organization of cells and functional properties of a tissue structure may be potentially useful for replacing defective tissues. Understanding how secreted signals from cells direct branching formation is important for engineering replacement tissue structures as well as repairing defective tissue structures. Matrix metalloproteinases (MMPs) have been identified previously as key mediators of branching morphogenesis presumably by degrading the extracellular matrix (ECM) in the fat pad. However, it is still unclear how these proteinases' signals provide spatially dependent patterning information to the branching tissue over time. I hypothesize that branching patterns are the result, at least in part, of proteinases' spatiotemporal activity profiles in the local microenvironment. MMPs have also been shown to play an important role in tumor progression by providing the proteolytic activity necessary for tumor cells to invade the ECM. It is reasonable to suspect that patterning information differs for branching in functionally normal tissues from that in malignant tissues, and these differences would be reflected in the proteinase signals' spatiotemporal activity profiles. To answer this question, methods for long term, real time, and quantitative measurement of secreted proteinases in the local microenvironment are needed but are currently lacking. Nanoplasmonic rulers consist of peptide- linked noble metal nanoparticles which can be used to quantitatively measure proteinase activity with single molecule sensitivity by optically monitoring their light scattering spectra over time. Nanoplasmonic rulers do not suffer from photobleaching or blinking (ie. fluctuating intensities), and therefore, nanoplasmonic rulers are capable of long term and continuous optical measurements of secreted proteinases. I propose to employ nanoplasmonic rulers, capable of long term, real time, and quantitative measurements of secreted proteinases, for the purpose of understanding the branching process and engineering branched structures. The following specific aims support the attainment of this goal: (1) Verify nanoplasmonic rulers are appropriate for long term, real time and quantitative measurements of proteinase activity using previously characterized nanoplasmonic rulers as a positive model of performance. (2) Identify spatiotemporal activity profiles of secreted proteinases during branching formation in non-malignant as compared to malignant organotypic cultures. (3) Determine whether modulation of proteases modifies spatiotemporal activity profiles and resulting branching patterns.